A crucial component in the ability of Mtb to survive in bactericidal environments such as the phagosome of macrophages is the efficient permeability barrier established by its unusual outer membrane (OM). We have discovered that an Mtb mutant lacking the OM channel protein Rv1698 was more susceptible to copper. Copper is an essential micronutrient, but excess copper is toxic. The intracellular copper concentration of the rv1698 mutant was 100-fold increased. Our experiments demonstrated that Rv1698 is required for copper efflux across the OM and for maintaining low intracellular copper levels. Because efflux against a concentration gradient requires energy, the OM component of efflux systems in E. coli is connected to an inner membrane pump which contributes substrate specificity and energy to the transport process. However, it is unknown how Mtb extrudes waste molecules, drugs, and other toxic solutes. Rv1698 is the first OM component of any efflux system in mycobacteria and, hence, represents a great opportunity not only to examine how Mtb controls essential, but toxic redox-active metals such as copper, but also efflux processes in Mtb in general. Copper inside Mtb-containing phagosomes is increased upon stimulation of macrophages with interferon-3 to concentrations that are sufficient to inhibit growth of Mtb indicating that copper homeostasis is critical for virulence of Mtb. However, this is a largely unexplored field for Mtb. We, therefore, propose to characterize the novel copper efflux channel MctB and interacting proteins, to identify missing components of copper homeostasis and to assess their role in virulence of Mtb. PUBLIC HEALTH RELEVANCE: Scientific interest in mycobacteria has not only been sparked by the paramount medical importance of Mycobacterium tuberculosis but also by its unusual outer membrane which is a crucial component in its ability to survive in bactericidal environments. We have discovered that a novel outer membrane channel protein of M. tuberculosis is required for efflux of copper. This protein is surface-accessible and represents the first outer membrane component of any efflux system in mycobacteria.